Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-05-03
2002-04-23
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023700, C536S024320, C435S810000
Reexamination Certificate
active
06376186
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to nucleic acid detection systems. More specifically, the invention relates to polynucleotide probes having binding specificity for rRNA or rDNA of bacteria that are members of the genus Staphylococcus.
BACKGROUND OF THE INVENTION
Bacteria among the genus Staphylococcus are classified as members of the broad Bacillus-Lactobacillus-Streptococcus cluster. The closest phylogenetic relatives of the genus Staphylococcus include the genera Bacillus, Bronchothrix, Enterococcus, Listeria and Planococcus. Staphylococcal bacteria are non-motile, gram-positive cocci 30 having genomic molar percentages of guanine and cytosine (G+C) in the range of from to 39%. These bacteria commonly are found on skin and mucosal surfaces of humans. Notably, these organisms can become opportunistic pathogens following trauma to the skin. Indeed,
S. aureus
is frequently associated with infections of the skin. Infections of wounds and deep tissue with this Staphylococcal species can become life-threatening.
It is well established that two single strands of deoxyribonucleic acid (“DNA”) or ribonucleic acid (“RNA”) can associate or “hybridize” with one another to form a double-stranded structure having two strands held together by hydrogen bonds between complementary base pairs. The individual strands of nucleic acid are formed from nucleotides that comprise the bases: adenine (A), cytosine (C), thymine (T), guanine (G), uracil (U) and inosine (I). In the double helical structure of nucleic acids, the base adenine hydrogen bonds with the base thymine or uracil, the base guanine hydrogen bonds with the base cytosine and the base inosine hydrogen bonds with adenine, cytosine or uracil. At any point along the chain, therefore, one may find the classical “Watson-Crick” base pairs A:T or A:U, T:A or U:A, and G:C or C:G. However, one may also find A:G, G:U and other “wobble” or mismatched base pairs in addition to the traditional (“canonical”) base pairs.
A double-stranded nucleic acid hybrid will result if a first single-stranded polynucleotide is contacted under hybridization-promoting conditions with a second single-stranded polynucleotide having a sufficient number of contiguous bases complementary to the sequence of the first polynucleotide. DNA/DNA, RNA/DNA or RNA/RNA hybrids may be formed under appropriate conditions.
Generally, a probe is a single-stranded polynucleotide having some degree of complementarity with the nucleic acid sequence that is to be detected (“target sequence”). Probes commonly are labeled with a detectable moiety such as a radioisotope, an antigen or a chemiluminescent moiety.
Descriptions of nucleic acid hybridization as a procedure for detecting particular nucleic acid sequences are given by Kohne in U.S. Pat. No. 4,851,330, and by Hogan et al., in U.S. Pat. Nos. 5,541,308 and 5,681,698. These references also describe methods for determining the presence of RNA-containing organisms in a sample which might contain such organisms. These procedures require probes that are sufficiently complementary to the ribosomal RNA (rRNA) of one or more non-viral organisms or groups of non-viral organisms. According to the method, nucleic acids from a sample to be tested and an appropriate probe are first mixed and then incubated under specified hybridization conditions. Conventionally, but not necessarily, the probe will be labeled with a detectable label. The resulting hybridization reaction is then assayed to detect and quantitate the amount of labeled probe that has formed duplex structures in order to detect the presence of rRNA contained in the test sample.
With the exception of viruses, all prokaryotic organisms contain rRNA genes encoding homologs of the procaryotic 5S, 16S and 23S rRNA molecules. In eucaryotes, these rRNA molecules are the 5S rRNA, 5.8S rRNA, 18S rRNA and 28S rRNA which are substantially similar to the prokaryotic molecules. Probes for detecting specifically targeted rRNA subsequences in particular organisms or groups of organisms in a sample have been described previously. These highly specific probe sequences advantageously do not cross react with nucleic acids from any other bacterial species or infectious agent under appropriate stringency conditions.
The present invention provides polynucleotide probes that can be used to detect the members of the genus Staphylococcus in a highly specific manner.
SUMMARY OF THE INVENTION
One aspect of the present invention relates to an oligonucleotide probe that specifically hybridizes a Staphylococcal nucleic acid target region corresponding to
E. coli
16S rRNA nucleotide positions 1276-1305 under a high stringency hybridization condition to form a detectable probe:target duplex. The oligonucleotide probe has a length of up to 100 nucleotides and includes at least 17 contiguous nucleotides contained within the sequence of SEQ ID NO:10. In a preferred embodiment, the oligonucleotide probe includes at least 30 contiguous nucleotides contained within the sequence of SEQ ID NO:10. The high stringency hybridization condition may be provided by either: (a) 0.48 M sodium phosphate buffer, 0. 1% sodium dodecyl sulfate, and 1 mM each of EDTA and EGTA, or (b) 0.6 M LiCl, 1% lithium lauryl sulfate, 60 mM lithium succinate and 10 mM each of EDTA and EGTA. The oligonucleotide probe may be made of DNA, but also may include at least one nucleotide analog. For example, the nucleotide analog may include a methoxy group at the 2′ position of a ribose moiety. In one embodiment the invented oligonucleotide probe has the sequence of any one of SEQ ID NO:1 or the complement thereof, SEQ ID NO:2 or the complement thereof, and SEQ ID NO:3 or the complement thereof. In a preferred embodiment, the sequence of the oligonucleotide is given by SEQ ID NO:2 or SEQ ID NO:3, and the oligonucleotide is a helper oligonucleotide. Any of the disclosed oligonucleotides can include a detectable label. Particular examples of detectable labels include chemiluminescent labels and radiolabels. In another preferred embodiment, the oligonucleotide probe has a sequence given by SEQ ID NO:1, and further includes a detectable label. A highly preferred detectable label is an acridinium ester.
Another aspect of the present invention relates to a probe composition for detecting nucleic acids of bacteria that are members of the Staphylococcus genus. This composition includes an oligonucleotide probe that hybridizes under a high stringency condition to a Staphylococcal target region corresponding to
E. coli
16S rRNA nucleotide positions 1276-1305 to form a detectable probe:target duplex. This oligonucleotide probe has a length of up to 100 nucleotide bases and includes at least 30 contiguous nucleotides contained within the sequence of SEQ ID NO:10 or the complement thereof. Under high stringency hybridization conditions the oligonucleotide probe specifically hybridizes nucleic acids present in
Staphylococcus aureus, Staphylococcus cohnii, Staphylococcus delphi, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus hyicus, Staphylococcus intermedius, Staphylococcus saprophyticus, Staphylococcus simulan
and
Staphylococcus warneri
. In certain embodiments, the oligonucleotide probe is made of DNA. Exemplary high stringency hybridization conditions are provided by either: (a) 0.48 M sodium phosphate buffer, 0.1% sodium dodecyl sulfate, and 1 mM each of EDTA and EGTA, or (b) 0.6 M LiCl, 1% lithium lauryl sulfate, 60 mM lithium succinate and 10 mM each of EDTA and EGTA. In a highly preferred embodiment, the oligonucleotide probe includes the sequence of SEQ ID NO:1 or the complement thereof. In another highly preferred embodiment, the length of the oligonucleotide probe is up to 60 bases. In and even more highly preferred embodiment of the invention, the oligonucleotide probe has the length and sequence of SEQ ID NO:1. Certain embodiments of the invented probe composition further include a detectable label on the oligonucleotide probe. For example, when the oligonucleotide prob
Gordon Patricia
Hogan James J.
Gen-Probe Incorporated
Gilly Michael J.
Johannsen Diana
Myers Carla J.
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